This invention relates to methods and compositions for sensitizing a cell to a compound such as a prodrug.
Working systems for in vivo human gene therapy are now established. Gene therapy vectors can be delivered to human cells in vivo by direct (local) injection or inhalation; by modified adenoviruses (reviewed in Englehardt, J. F. xe2x80x9cMethods for Adenovirus-Mediated Gene Transfer to Airway Epitheliumxe2x80x9d Chapter 11 in Methods in Molecular Medicine, Gene Therapy Protocols, Ed. P. Robbins, 1997, Humana Press Inc., Totowa, N.J.); by retroviruses (Olsen, J. C., et al., xe2x80x9cMethods for the Use of Retroviral Vectors for Transfer of the CFTR Gene to Airway Epithelium,xe2x80x9d Chapter 10, Ibid.); by cationic lipid-plasmid aggregates (Nabel, G. J., et al., xe2x80x9cMethods for Liposome-Mediated Gene Transfer to Tumor Cells in Vivo,xe2x80x9d Chapter 21, Ibid.; Son, K., et al., xe2x80x9cCationic Liposome-Mediated Gene Transfer to Tumor Cells in Vitro and In Vivo,xe2x80x9d Chapter 23, Ibid.); or, simply, by delivery of naked DNA (see, U.S. Pat. No. 5,589,466 to Felgner, et al.).
Systemic delivery for in vivo gene therapy, i.e., delivery of the vector to a distal target cell via body systems such as the circulation, which is a less well explored avenue, has been achieved using lipid-plasmid particles such as those disclosed in published PCT patent application WO 96/40964, U.S. Pat. No. 5,705,385, and U.S. patent applications Ser. Nos. 08/485,458, 08/484,282, 08/660,025, 08/856,374, 60/063,473 and 09/169,573, filed Oct. 9, 1998, all of which are assigned to the assignee of the instant invention and incorporated herein by reference. This latter format provides a fully encapsulated lipid-plasmid particle that protects the vector from nuclease degradation in serum, is non-immunogenic, is small in size and has a prolonged circulation half-life.
A variation of the basic gene therapy technique that is useful for therapeutic treatment is gene-delivered enzyme prodrug therapy (xe2x80x9cGDEPTxe2x80x9d). GDEPT is also known as the xe2x80x9csuicide gene/prodrugxe2x80x9d system and was first developed by Moolten, F. L., Cancer Res. 46:5276-5281 (1986). In addition, for a detailed review of GDEPT, see, Moolten, F. L., Chapter 11 (1995), The Internet Book Of Gene Therapy, Cancer Therapeutics, Eds. Sobol, R. E., Scanlon, K. J., Appelton and Lange. In this method, a heterologous gene, encoding an enzyme that promotes the metabolism of a first compound, to which the cell is less sensitive (i.e., the xe2x80x9cprodrugxe2x80x9d), into a second compound to which is cell is more sensitive, is delivered to a cell. The cell takes up the gene and expresses it. Then the prodrug is delivered to the cell. The enzyme will process the prodrug into the second compound, and respond accordingly. A suitable system proposed by Moolten is the herpes simplex virusxe2x80x94thymidine kinase (HSV-TK) gene, and the prodrug ganciclovir. This method has recently been employed in work such as that of Zerrouqui, et al., Cancer. Gen. Therapy 3(6):385-392 (1996). Cationic lipid-nucleic aggregates were used for local delivery of the TK gene to mouse tumors in Sugaya, S., et al., Hum. Gen. Ther. 7:223-230 (1996). Human clinical trials using a GDEPT system employing viral vectors have been proposed (see, Hum. Gene Ther. 8:597-613 (1997) and Hum. Gen. Ther. 7:255-267 (1996)).
Patent applications relating to the GDEPT method have been published under the following numbers: WO 97/19180; WO 97/07118; WO 96/22277; WO 97/19183; WO 96/16179; WO 96/03515; WO 96/03515; WO 96/03151; EP 690129; EP 657541; EP 657539; WO 95/05835 and EP 415731.
Prior art methods suffer from many deficiencies. Firstly, the vector systems employed to date in GDEPT are designed for local delivery of the vector only. These systems are also highly immunogenic and hinder repeat dosing. This limits the range of applications for GDEPT. Secondly, non-specific toxicity of the prodrug can prohibit delivery of a satisfactory amount of the prodrug for effecting the transformed cell. In addition, prior art prodrug formulations are rapidly cleared from the blood, thereby requiring less desirable treatment modalities such as repeat injections or intravenous drip.
Another deficiency with the prior art methods is that even though researchers struggle to improve gene delivery systems, virtually no work has explored improvements in the prodrug delivery system, or the possible advantages of syncopating methods of delivering prodrugs and the vector. If the gene vector element is delivered in a format different from the prodrug element (i.e., adenovirus delivered vector versus free drug), the biodistribution of the elements is different, thus complicating dosage requirements and associated toxicities.
Clearly, there remains a need in the art for a method that solves the problem of targeting both elements of the suicide gene/prodrug system to the same organ, tumor or disease site of interest. It would be advantageous if this method could also reduce non-specific toxicities of the suicide gene/prodrug elements and extend their half-life in blood. The present invention fulfills these and other needs.
The present invention provides, inter alia, methods and compositions for sensitizing a cell to a compound, such as a prodrug. In one embodiment, the present invention provides a method of sensitizing a cell to a compound, the method comprising: (a) transfecting a cell with a vector comprising a gene sequence encoding a gene-product that promotes the processing, i.e., conversion, of a first compound (e.g., a prodrug) into a second compound; and (b) delivering to the cell the first compound in a lipid formulation; wherein the cell is more sensitive to the second compound than to the first compound.
In the above method, both the vector and the first compound can be delivered in lipid formulations which can be the same or different. The lipid formulations, whether used to deliver the vector or first compound (e.g., prodrug), can be prepared from a variety of lipids, lipid conjugates and additional compatible components known in the art. The lipid formulations can be prepared, for example, from sphingomyelin and cholesterol. Moreover, the lipid formulations can contain additional components which improve the properties or characteristics of the formulations, such as leakiness, longevity in circulation, reduced toxicity, encapsulation efficiency, etc. Such components include, for example, cationic lipids, ATTA-lipid conjugates, PEG-lipid conjugates, targeting agents, etc. Once prepared, the lipid formulations can be administered or delivered to the cell using a variety of techniques known to those of skill in the art. For instance, the lipid formulations can be delivered systemically, regionally or locally.
In a preferred embodiment, the vector is also delivered in a lipid formulation, such as a lipid-encapsulated formulation that is prepared for in vivo administration. In another preferred embodiment, the vector is a cationic lipid-vector aggregate or particle. The vector can be, for example, a modified adenovirus, modified retrovirus or naked DNA. The gene-product can be any product which promotes the processing, i.e., conversion, of a first compound (e.g., a prodrug) into a second compound to which the cell of interest is sensitive or receptive. Examples of suitable gene-products include, but are not limited to, herpes simplex virus thymidine kinase, cytosine deaminase, xanthine-guaninephosphoribosyl transferase, purine nucleoside phosphorylase, cytochrome P450 2B1 and their analogs. Other gene products suitable for use in the methods of the present invention will be readily apparent to those of skill in the art.
In a preferred embodiment, the first compound is a prodrug, i.e., a compound to which the cell of interest in not initially sensitive to, but which the gene-product converts into a compound to which the cell of interest is sensitive. Examples of suitable prodrugs include, but are not limited to, ganciclovir, acyclovir, bromovinyldeoxyuridine, 5-fluorocytosine, 6-thioxanthine, MeP-dr and cyclophosphamide. Other prodrugs suitable for use in the methods of the present invention will be readily apparent to those of skill in the art.
In another embodiment, the present invention provides a method of sensitizing a cell to a compound, the method comprising: a) delivering to a cell an enzyme which promotes the processing of a first compound into a second compound; and b) delivering to the cell the first compound in a lipid formulation; wherein the cell is more sensitive to the second compound than the first compound. In a presently preferred embodiment, both the enzyme and the first compound are delivered in lipid formulations.
In yet another embodiment, the present invention provides a composition for treating a human condition (e.g., a human medical disorder or disease state), the composition comprising a prodrug in a lipid formulation and a pharmaceutically acceptable carrier. In a presently preferred embodiment, the composition further comprises a vector or an enzyme in a lipid formulation.
In still another embodiment, the present invention provides a kit for the treatment of a human medical disorder, the kit comprising: a) a vector in a lipid formulation; and b) a prodrug in a lipid formulation.
In addition, the present invention provides methods for preparing lipid formulated prodrugs, vectors and enzymes which can be used in carrying out the methods of the present invention.
Other features, objects and advantages of the invention and its preferred embodiments will become apparent from the detailed description which follows.